It is now well established that angiogenesis is implicated in the pathogenesis of a variety of disorders. These include solid tumors and metastasis, atherosclerosis, retrolental fibroplasia, hemangiomas, chronic inflammation, intraocular neovascular diseases such as proliferative retinopathies, e.g., diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma, immune rejection of transplanted corneal tissue and other tissues, rheumatoid arthritis, and psoriasis. Folkman et al., J. Biol. Chem., 267:10931-10934 (1992); Klagsbrun et al., Annu. Rev. Physiol. 53:217-239 (1991); and Garner A., “Vascular diseases”, In: Pathobiology of Ocular Disease. A Dynamic Approach, Garner A., Klintworth G K, eds., 2nd Edition (Marcel Dekker, NY, 1994), pp 1625-1710.
In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia, and for providing nourishment for the growth and metastasis of the tumor. Folkman et al., Nature 339:58 (1989). The neovascularization allows the tumor cells to acquire a growth advantage and proliferative autonomy compared to the normal cells. A tumor usually begins as a single aberrant cell which can proliferate only to a size of a few cubic millimeters due to the distance from available capillary beds, and it can stay ‘dormant’ without further growth and dissemination for a long period of time. Some tumor cells then switch to the angiogenic phenotype to activate endothelial cells, which proliferate and mature into new capillary blood vessels. These newly formed blood vessels not only allow for continued growth of the primary tumor, but also for the dissemination and recolonization of metastatic tumor cells. Accordingly, a correlation has been observed between density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors. Weidner et al., N Engl. J. Med 324:1-6 (1991); Horak et al., Lancet 340:1120-1124 (1992); Macchiarini et al., Lancet 340:145-146 (1992). The precise mechanisms that control the angiogenic switch is not well understood, but it is believed that neovascularization of tumor mass results from the net balance of a multitude of angiogenesis stimulators and inhibitors (Folkman Nat Med 1(1):27-31 (1995)).
It is currently accepted that metastases are responsible for the vast majority, estimated at 90%, of deaths from solid tumors (Gupta and Massague, Cell 127, 679-695 (2006)). The complex process of metastasis involves a series of distinct steps including detachment of tumor cells from the primary tumor, intravasation of tumor cells into lymphatic or blood vessels, and extravasation and growth of tumor cells in secondary sites. Analysis of regional lymph nodes in many tumor types suggests that the lymphatic vasculature is an important route for the dissemination of human cancers. Furthermore, in almost all carcinomas, the presence of tumor cells in lymph nodes is the most important adverse prognostic factor. While it was previously thought that such metastases exclusively involved passage of malignant cells along pre-existing lymphatic vessels near tumors, recent experimental studies and clinicopathological reports (reviewed in Achen et al., Br J Cancer 94 (2006), 1355-1360 and Nathanson, Cancer 98, 413-423 (2003)) suggest that lymphangiogenesis can be induced by solid tumors and can promote tumor spread. These and other recent studies suggest targeting lymphatics and lymphangiogenesis may be a useful therapeutic strategy to restrict the development of cancer metastasis, which would have a significant benefit for many patients.
VEGFC, a member of the vascular endothelial cell factor (VEGF) family, is one of the best studied mediators of lymphatic development. Overexpression of VEGFC in tumor cells was shown to promote tumor-associated lymphangiogenesis, resulting in enhanced metastasis to regional lymph nodes (Karpanen et al., Faseb J 20, 1462-1472 (2001); Mandriota et al., EMBO J 20, 672-682 (2001); Skobe et al., Nat Med 7, 192-198 (2001); Stacker et al., Nat Rev Cancer 2, 573-583 (2002); Stacker et al., Faseb J 16, 922-934 (2002)). VEGFC expression has also been correlated with tumor-associated lymphangiogenesis and lymph node metastasis for a number of human cancers (reviewed in Achen et al., 2006, supra. In addition, blockade of VEGFC-mediated signaling has been shown to suppress tumor lymphangiogenesis and lymph node metastases in mice (Chen et al., Cancer Res 65, 9004-9011 (2005); He et al., J. Natl Cancer Inst 94, 8190825 (2002); Krishnan et al., Cancer Res 63, 713-722 (2003); Lin et al., Cancer Res 65, 6901-6909 (2005)).
VEGFC is known to bind at least two cell surface receptor families, the tyrosine kinase VEGF receptors and the neuropilin (Nrp) receptors.
Of the three VEGF receptors, VEGFC can bind VEGFR2 and VEGFR3 leading to receptor dimerization (Shinkai et al., J Biol Chem 273, 31283-31288 (1998)), kinase activation and autophosphorylation (Heldin, Cell 80, 213-223 (1995); Waltenberger et al., J. Biol Chem 269, 26988-26995 (1994)). The phosphorylated receptor induces the activation of multiple substrates leading to angiogenesis and lymphangiogenesis (Ferrara et al., Nat Med 9, 669-676 (2003)).
The neuropilin (Nrp) family is comprised of two homologous proteins, neuropilin-1 (Nrp1) and neuropilin-2 (Nrp2). In addition to the VEGF receptors, VEGFC also binds to Nrp2, which was initially identified as class 3 semaphorin receptor and mediator of axon guidance (Favier et al., Blood 108, 1243-1250 (2006); Soker et al., J Cell Biochem 85, 357-368 (2002)). Multiple lines of evidence implicate Nrp2 in the development of the vascular and lymphatic systems. Homozygous Nrp2 mutants show a severe reduction of small lymphatic vessels and capillaries prenatally (Yuan et al., Development 129, 4797-4806 (2002)). Furthermore, the dramatic and embryonic lethal vascular defect seen in homozygous Nrp1 mutant mice is enhanced by loss of Nrp2 function leading to earlier lethality (Takashima et al., Proc Natl Acad Sci USA 99, 3657-3662 (2002)). However, the role of Nrp2 in modulating adult vascular and lymphatic biology, and more specifically metastasis is unknown.
Nrps have short intracellular domains that are not known to have any enzymatic or signaling activity. It has been proposed that Nrps function to enhance VEGFR signaling by enhancing ligand-VEGF receptor binding (Favier et al., 2006, supra; Soker et al., 2002, supra). Additionally, sema3F, the semaphorin ligand of Nrp2, has been shown to modulate endothelial cell behavior in vitro and in vivo (Bielenberg et al., J Clin Invest 114, 1260-1271 (2004); Favier et al., Blood 1243-1250, (2006)). However, recent reports have suggested an alternate possibility that Nrps may function independently of VEGF receptors or semaphorin function to modulate endothelial cell (EC) migration (Murga et al., Blood 105, 1992-1999 (2005); Pan et al., Cancer Cell 11, 53-67 (2007); Wang et al., J Biol Chem 278, 48848-48860 (2003)).
Anti-VEGF neutralizing antibodies suppress the growth of a variety of human tumor cell lines in nude mice (Kim et al., Nature 362:841-844 (1993); Warren et al., J. Clin. Invest. 95:1789-1797 (1995); Borgström et al., Cancer Res. 56:4032-4039 (1996); Melnyk et al., Cancer Res. 56:921-924 (1996)) and also inhibit intraocular angiogenesis in models of ischemic retinal disorders. Adamis et al., Arch. Ophthalmol. 114:66-71 (1996). Therefore, anti-VEGF monoclonal antibodies or other inhibitors of VEGF action are promising candidates for the treatment of tumors and various intraocular neovascular disorders. Such antibodies are described, for example, in EP 817,648 published Jan. 14, 1998; and in WO98/45331 and WO98/45332, both published Oct. 15, 1998. One of the anti-VEGF antibodies, bevacizumab, has been approved by the FDA for use in combination with a chemotherapy regimen to treat metastatic colorectal cancer (CRC) and non-samll cell lung cancer (NSCLC). And bevacizumab is being investigated in many ongoing clinical trials for treating various cancer indications.
Other anti-VEGF antibodies and anti-Nrp1 antibodies are also known, and described, for example, in Liang et al., J Mol Biol 366, 815-829 (2007); Pan et al., Cancer Cell 11, 53-67 (2007; and Liang et al., J Biol Chem 281, 951-961 (2006)).